Despite its frequent use as an anticoagulant, the actual mechanism by which heparin dramatically increases the anticoagulant activity of antithrombin III is not well understood. Using site-directed mutagenesis techniques and an expression system which produces physically and functionally homogeneous ATIII, some of the important ionic interactions in ATIII heparin binding have been identified. On the basis of this information and knowledge of protein conformational changes that occur during proteinase inhibition by serpins, a working model for the molecular mechanism underlying ATIII heparin cofactor activity has been proposed. It is hypothesized that by providing pairing alternatives for positively charged residues that participate in fixing the native conformation of ATIII and by inducing rotation of a critically located arginine residue in sheet A, heparin binding to ATIII promotes rearrangement and movement of the s123A-hDEF fragment. This opens the A sheet so that the reactive loop can become partially inserted into sheet A, generating an activated conformation which is primed for inhibition of ATIII's target proteinases. The goal of work proposed in this application is to elucidate the structural basis of ATIII heparin cofactor activity. Specific Aim 1 is to definitively identify the heparin and pentasaccharide binding sites on ATIII by measuring heparin binding site variant heparin and pentasaccharide affinities and docking the pentasaccharide on antithrombin in molecular modeling studies. Specific Aim 2 is to obtain detailed information on the roles of individual heparin binding residues in the process of heparin cofactor activation by determining kinetic constants for the interactions of variants with heparins and proteinases. Specific Aim 3 is to investigate contributions of nonbasic residues to heparin cofactor activity. These studies will focus on: (i) the roles of several negatively charged residues which are proposed to salt bridge with positively charged heparin binding residues in native ATIII; (ii) tryptophan 189, which is believed to be the reporter group for heparin induced fluorescence enhancement of ATIII; and (iii) binding site aromatic and hydrogen bonding residues which have the potential to interact with heparin.